The preparation of polypeptides using recombinant technology has developed into a standard procedure during the last couple of decades. The access to recombinant polypeptides by cloning the genes encoding the respective polypeptide followed by subsequent transformation of suitable expression hosts with the gene to be expressed and final production and purification of the obtained recombinant polypeptide product has provided access to a whole new class of biologically designed and produced therapeutics.
Pharmaceutically active compounds have been prepared in increasing numbers in the pharmaceutical industry using recombinant DNA technology followed by production processes developed in the field of bioengineering.
Such biological products include monoclonal antibodies, which have been developed into important treatment options in various medical fields including autoimmune diseases, inflammatory disorders, immunosuppression oncology or the like.
Development of such therapeutics of biological origin requires production at industrial scale thereby providing access to large amounts of recombinant polypeptide. Preferred expression systems are mammalian cell cultures which are superior to most other eukaryotic systems based on insect cells, yeast or the like, or even traditional prokaryotic expression systems.
However, mammalian cell culture includes tremendous challenges especially at the industrial scale. Production facilities for mammalian cell culture require thorough optimization of many process conditions.
One of the most important process parameters for controlling the overall production process is the medium in which cells are grown and polypeptide production takes place. Suitable cell culture media must provide cell cultures with all necessary nutrient substances, which is especially difficult if no components of animal origin like serum or proteins, e.g. growth factors, are added to the media.
Consequently, a great variety of different cell culture media have been developed. In some cases, the focus has been on the general composition and media with a large variety of different substances have been proposed (U.S. Pat. No. 5,122,469, EP 0 481 791, EP 0 283 942). In other cases, particular ingredients have been suggested to improve cell culture. Major goals have been to improve either growth or survival of the cells, or the quantity and quality of recombinantly expressed polypeptides.
Specific aspects addressed in prior art documents are amongst others the contribution of particular trace ions (e.g. WO 02/066603, EP 0 872 487, EP 1 360 314 A2), vitamins such as ascorbic acid (e.g. U.S. Pat. No. 6,838,284), carbohydrates (EP 1 543 106) or the content of specific amino acids in combination with additional features (e.g. EP 0 501 435, U.S. Pat. No. 5,830,761, U.S. Pt. No. 7,294,484).
The major ions and their concentrations in cell culture media are largely kept constant and remain unconsidered and unchanged. All classical types of media such as e.g. DMEM, DMEM/F12. BME or RPMI 1640 use relatively narrow and fixed ranges for the concentrations of bulk ions in general and the monovalent cations Na+ and K+ in particular. This is in line with the fact that the ionic balance of the bulk ions in general and the monovalent cations Na+ and K+ in particular is a rather universal property of almost all mammalian cells.
In more detail, the transmembrane gradient of sodium and potassium ions is a basic property of mammalian cells with high concentration of potassium ions inside the cell and high concentration of sodium ions outside the cell. The sodium potassium pump is one of the major ion pumps of the cell membrane which is electrogenic and contributes in establishing and maintaining the respective sodium and potassium ionic gradient across the membrane (Kaplan, Membrane cation transport and the control of proliferation of mammalian cells. Annu Rev Physiol.; 40: 19-41 (1978)). The pump uses about 30% of the cells energy and is one of the major energy consuming processes of the cells. Many basic biochemical processes are coupled to the electrochemical gradient of sodium ions, such as e.g. the Na+/Ca+ exchanger or the amino acid transport into cells. The concentrations of sodium and potassium ions outside a cell are therefore parameters of paramount importance that influence the gradient of these ions across the membrane and the basic status of the cell.
In accordance with the typical concentration of sodium ions inside and outside a generic mammalian cell (Alberts et al., Molecular Biology of the Cell (1994)) mostly sodium concentrations of about 145 mM are chosen together with potassium ion concentrations of around 5 mM. For most media types this results in a ratio between sodium and potassium ions that ranges between about 20-30 (see Table 1 below and e.g. U.S. Pat. No. 5,135,866).
Only few prior art documents describe cell culture media suitable for mammalian cell culture or the production of recombinant proteins mentioning specific ratios of sodium to potassium ions. These documents suggest media compositions with specifically high ratios in the high range of about 30.7 in U.S. Pat. No. 5,232,848, or in a range of between about 25 and 35 thus reaching even higher values (EP 0 283 942, EP 0 389 786). Other media such as HAM's-F12 or defined animal cell culture media as proposed in US 2008/0261259 also specifically suggest higher values (e.g. 27.9 to 57.5 in US 2008/0261259). Only very few documents disclose media having a ratio of sodium to potassium ions below 20, such as 11.5-30 (U.S. Pat. No. 7,294,484) or a ratio of about 15 (U.S. Pat. No. 6,180,401). These documents still use ratios of higher than 10 and also do not assign a particular advantage to changing this parameter to the values as mentioned.
In addition to the effects related to the ionic balance between particular ions, also the contribution of the major ions to the overall osmolality of the medium has to be considered. Most conventional media such as e.g. DMEM, MEM alpha, or Fischer's medium are characterized by a high amount of sodium chloride.
WO 02/101019 addresses the high content of glucose in the medium in combination with usage of a higher osmolality. The high glucose concentration between about 2-40 has been achieved by reducing or even completely eliminating agents such as sodium chloride thereby maintaining osmolality at a given level.
Considering the above challenges and existing disadvantages, there is a continued need in the field of industrial biotechnology for improved cell culture media which allow producing recombinant polypeptides at an industrial scale.